Project description:Human embryonic stem cells (WiCell H9 passage 52) were differentiated following the Novocell protocol (D'Amour et al. Nature Biotechnology, 2005). The two biological replicates (Trial 15 and Trial 16) were analyzed by Real-Time PCR utilizing TaqMan Low Density Array Cards that have been customized for a set of 46 genes specific for endoderm and pancreatic gene expression.
Project description:PeptideRank is an approach that uses a rank-based algorithm for peptide detectability prediction from shotgun proteomics data. The best performance is achieved when it is trained on organism-specific shotgun proteomics datasets. The Drosophila shotgun proteomics data presented here have been used amongst others to train and validate PeptideRank.
Project description:PeptideRank is an approach that uses a rank-based algorithm for peptide detectability prediction from shotgun proteomics data. The best performance is achieved when it is trained on organism-specific shotgun proteomics datasets. The Drosophila shotgun proteomics data presented here have been used amongst others to train and validate PeptideRank.
Project description:The goal of bone tissue engineering is to build artificial bone tissue with properties that closely resemble human bone and thereby support the optimal integration of the constructs (biografts) into the body. The development of tissues in 3D scaffolds includes several complex steps that need to be optimized and monitored. In particular, cell-material interaction during seeding, cell proliferation and cell differentiation within the scaffold pores play a key role. In this work, we seeded two types of 3D-printed scaffolds with pre-osteoblastic MC3T3-E1 cells, proliferated and differentiated the cells, before testing and adapting different assays and imaging methods to monitor these processes. Alpha-TCP/HA (α-TCP with low calcium hydroxyapatite) and baghdadite (Ca3ZrSi2O9) scaffolds were used, which had comparable porosity (~50%) and pore sizes (~300-400 µm). Cell adhesion to both scaffolds showed ~95% seeding efficiency. Cell proliferation tests provided characteristic progression curves over time and increased values for α-TCP/HA. Transmitted light imaging displayed a homogeneous population of scaffold pores and allowed us to track their opening state for the supply of the inner scaffold regions by diffusion. Fluorescence labeling enabled us to image the arrangement and morphology of the cells within the pores. During three weeks of osteogenesis, ALP activity increased sharply in both scaffolds, but was again markedly increased in α-TCP/HA scaffolds. Multiphoton SHG and autofluorescence imaging were used to investigate the distribution, morphology, and arrangement of cells; collagen-I fiber networks; and hydroxyapatite crystals. The collagen-I networks became denser and more structured during osteogenic differentiation and appeared comparable in both scaffolds. However, imaging of the HA crystals showed a different morphology between the two scaffolds and appeared to arrange in the α-TCP/HA scaffolds along collagen-I fibers. ALP activity and SHG imaging indicated a pronounced osteo-inductive effect of baghdadite. This study describes a series of methods, in particular multiphoton imaging and complementary biochemical assays, to validly measure and track the development of bone tissue in 3D scaffolds. The results contribute to the understanding of cell colonization, growth, and differentiation, emphasizing the importance of optimal media supply of the inner scaffold regions.
Project description:The nematode pharynx has a potassium channel with unusual properties, which allows the muscles to repolarize quickly and with the proper delay. Here, the Caenorhabditis elegans exp-2 gene is shown to encode this channel. EXP-2 is a Kv-type (voltage-activated) potassium channel that has inward-rectifying properties resembling those of the structurally dissimilar human ether-à-go-go-related gene (HERG) channel. Null and gain-of-function mutations affect pharyngeal muscle excitability in ways that are consistent with the electrophysiological behavior of the channel, and thereby demonstrate a direct link between the kinetics of this unusual channel and behavior.
Project description:Myotonic dystrophy type 1 (DM1) is caused by an expanded CUG repeat (CUG(exp)) that sequesters muscleblind-like 1 protein (MBNL1), a protein that regulates alternative splicing. CUG(exp) RNA is a validated drug target for this currently untreatable disease. Herein, we develop a bioactive small molecule (1) that targets CUG(exp) RNA and is able to inhibit the CUG(exp)·MBNL1 interaction in cells that model DM1. The core of this small molecule is based on ligand 2, which was previously reported to be active in an in vitro assay. A polyamine-derivative side chain was conjugated to this core to make it aqueous-soluble and cell-penetrable. In a DM1 cell model this conjugate was found to disperse CUG(exp) ribonuclear foci, release MBNL1, and partially reverse the mis-splicing of the insulin receptor pre-mRNA. Direct evidence for ribonuclear foci dispersion by this ligand was obtained in a live DM1 cell model using time-lapse confocal microscopy.
Project description:With multiple groups generating methods for differentiating induced pluripotent stem cells (hiPSCs) into endothelial cells/endothelial progenitors (ECs/EPs), there is a need to better understand the specific endothelial subtypes that different differentiation protocols produce. This is especially important for accurate tissue modeling as researchers continue to incorporate the endothelium into engineered tissues. We illustrated the heterogeneity of cells produced from different differentiation protocols by focusing on two selected protocols, one driving differentiation exclusively using small molecules (SM Protocol) and one driving differentiation primarily using growth factors (GF Protocol). We characterized the cells through a combination of vasculogenic computational analysis following encapsulation in 3D hydrogels, RNA sequencing, and measurement of soluble factor release. Vasculogenic computational analysis indicates that cells from the GF protocol formed more dense and interconnected vasculature compared to cells from the SM protocol. Likewise, RNA-seq analysis showed GF-derived cell enrichment in pathways involved in cell migration and angiogenesis. In addition, GF-derived cells favor differentiation to arterial endothelial cells as well as predisposition to undergoing a partial endothelial-to-mesenchymal transition, whereas SM-derived cells resemble immature progenitors based on higher proliferation rates as well as ECM remodeling. These trends also persisted following extended 3D culture. The results demonstrate that despite using the same starting hiPSC population and isolating EPs using the same surface marker, the two differentiation protocols yield highly distinct cell populations. This work highlights the importance of understanding the specific endothelial subtypes produced by different differentiation protocols for the creation of more accurate tissue models.
Project description:The term "retroactive avoidance" refers to a special class of effects of future stimulus presentations on past behavioral responses. Specifically, it refers to the anticipatory avoidance of aversive stimuli that were unpredictable through random selection after the response. This phenomenon is supposed to challenge the common view of the arrow of time and the direction of causality. Preliminary evidence of "retroactive avoidance" has been published in mainstream psychological journals and started a heated debate about the robustness and the true existence of this effect. A series of seven experiments published in 2014 in the Journal of Consciousness Studies (Maier et al., 2014) tested the influence of randomly drawn future negative picture presentations on avoidance responses based on key presses preceding them. The final study in that series used a sophisticated quantum-based random stimulus selection procedure and implemented the most severe test of retroactive avoidance within this series. Evidence for the effect, though significant, was meager and anecdotal, Bayes factor (BF10) = 2. The research presented here represents an attempt to exactly replicate the original effect with a high-power (N = 2004) preregistered multi-lab study. The results indicate that the data favored the null effect (i.e., absence of retroactive avoidance) with a BF01 = 4.38. Given the empirical strengths of the study, namely its preregistration, multi-lab approach, high power, and Bayesian analysis used, this failed replication questions the validity and robustness of the original findings. Not reaching a decisive level of Bayesian evidence and not including skeptical researchers may be considered limitations of this study. Exploratory analyses of the change in evidence for the effect across time, performed on a post-hoc basis, revealed several potentially interesting anomalies in the data that might guide future research in this area.
Project description:Osteoblastic mineralization occurs during the early stages of bone formation. During this mineralization, hydroxyapatite (HA), a major component of bone, is synthesized, generating hard tissue. Many of the mechanisms driving biomineralization remain unclear because the traditional biochemical assays used to investigate them are destructive techniques incompatible with viable cells. To determine the temporal changes in mineralization-related biomolecules at mineralization spots, we performed time-lapse Raman imaging of mouse osteoblasts at a subcellular resolution throughout the mineralization process. Raman imaging enabled us to analyze the dynamics of the related biomolecules at mineralization spots throughout the entire process of mineralization. Here, we stimulated KUSA-A1 cells to differentiate into osteoblasts and conducted time-lapse Raman imaging on them every 4 hours for 24 hours, beginning 5 days after the stimulation. The HA and cytochrome c Raman bands were used as markers for osteoblastic mineralization and apoptosis. From the Raman images successfully acquired throughout the mineralization process, we found that β-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization. A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization. We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable.